Polypeptides With Enhanced Anti-Inflammatory And Decreased Cytotoxic Properties And Relating Methods

ABSTRACT

The invention provides a polypeptide containing at least one IgG Fc region, wherein said at least one IgG Fc region is glycosylated with at least one galactose moiety connected to a respective terminal sialic acid moiety by a α2,6 linkage, and wherein said polypeptide having a higher anti-inflammatory activity as compared to an unpurified antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of patent application Ser. No.12/013,212, filed on Jan. 11, 2008, which is a continuation of patentapplication Ser. No. 11/957,015, filed on Dec. 14, 2007, which claimsthe benefit of continuation-in-part patent application of PCT PatentApplication Number PCT/US 07/08396, filed on Apr. 3, 2007, which claimsthe benefit of U.S. Provisional Patent Application No. 60/789,384, filedon Apr. 5, 2006, all of which are incorporated herein by reference.Patent application Ser. No. 12/013,212 is also a continuation patentapplication of PCT Patent Application Number PCT/US07/72771 filed onJul. 3, 2007, which is also incorporated herein by reference.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

The Research leading to the present invention was supported in part, byNational Institutes of Health Grant No. AI 034662. Accordingly, the U.S.Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to a novel method for designingtherapeutic polypeptides for treatment of inflammatory diseases.

BACKGROUND

Although cellular receptors for immunoglobulins were first identifiednearly 40 years ago, their central role in the immune response was onlydiscovered in the last decade. They are key players in both the afferentand efferent phase of an immune response, setting thresholds for B cellactivation and antibody production, regulating the maturation ofdendritic cells and coupling the exquisite specificity of the antibodyresponse to effector pathways, such as phagocytosis, antibody dependentcellular cytotoxicity and the recruitment and activation of inflammatorycells. Their central role in linking the humoral immune system to innateeffector cells has made them attractive immunotherapeutic targets foreither enhancing or restricting the activity of antibodies in vivo.

The interaction of antibodies and antibody-antigen complexes with cellsof the immune system effects a variety of responses, including antibodydependent cell-mediated cytotoxicity (ADCC) and complement dependentcytotoxicity (CDC), phagocytosis, inflammatory mediator release,clearance of antigen, and antibody half-life (reviewed in Daron, AnnuRev Immunol, 15, 203-234 (1997); Ward and Ghetie, Therapeutic Immunol,2, 77-94 (1995); Ravetch and Kinet, Annu Rev Immunol, 9, 457-492(1991)), each of which is incorporated herein by reference).

Antibody constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions.Depending on the amino acid sequence of the constant region of theirheavy chains, antibodies or immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2.The heavy chain constant regions that correspond to the differentclasses of immunoglobulins are called α, δ, ε, γ, and μ, respectively.Of the various human immunoglobulin classes, human IgG1 and IgG3 mediateADCC more effectively than IgG2 and IgG4.

Papain digestion of antibodies produces two identical antigen bindingfragments, called Fab fragments, each with a single antigen bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. The Fc region is central to the effector functionsof antibodies. The crystal structure of the human IgG Fc region has beendetermined (Deisenhofer, Biochemistry, 20, 2361-2370 (1981), which isincorporated herein by reference). In human IgG molecules, the Fc regionis generated by papain cleavage N-terminal to Cys, 226.

IgG has long been appreciated to mediate both pro- and anti-inflammatoryactivities through interactions mediated by its Fc fragment. Thus, whileFc-FcyR interactions are responsible for the pro-inflammatory propertiesof immune complexes and cytotoxic antibodies, intravenous gamma globulin(IVIG) and its Fc fragments are anti-inflammatory and are widely used tosuppress inflammatory diseases. The precise mechanism of suchparadoxical properties is unclear but it has been proposed thatglycosylation of IgG is crucial for regulation of cytotoxicity andinflammatory potential of IgG.

IgG contains a single, N-linked glycan at Asn²⁹⁷ in the CH2 domain oneach of its two heavy chains. The covalently-linked, complexcarbohydrate is composed of a core, biantennary penta-polysaccharidecontaining N-acetylglucosamine (GIcNAc) and mannose (man). Furthermodification of the core carbohydrate structure is observed in serumantibodies with the presence of fucose, branching GIcNAc, galactose(gal) and terminal sialic acid (sa) moieties variably found. Over 40different glycoforms have thus been detected to be covalently attachedto this single glycosylation site. Fujii et al., J. Biol. Chem 265, 6009(1990). Glycosylation of IgG has been shown to be essential for bindingto all FcyRs by maintaining an open conformation of the two heavychains. Jefferis and Lund, Immune.l Lett. 82, 57 (2002), Sondermann etal., J. Mol. Biol. 309, 737 (2001). This absolute requirement of IgGglycosylation for FcyR binding accounts for the inability ofdeglycosylated IgG antibodies to mediate in vivo triggered inflammatoryresponses, such as ADCC, phagocytosis and the release of inflammatorymediators. Nimmerjahn and Ravetch, Immunity 24, 19 (2006). Furtherobservations that individual glycoforms of IgG may contribute tomodulating inflammatory responses has been suggested by the alteredaffinities for individual FcyRs reported for IgG antibodies containingor lacking fucose and their consequential affects on cytotoxicity.Shields et al., J. Biol. Chem. 277, 26733 (2002), Nimmerjahn andRavetch, Science 310, 1510 (2005). A link between autoimmune states andspecific glycosylation patterns of IgG antibodies has been observed inpatients with rheumatoid arthritis and several autoimmune vasculities inwhich decreased galactosylation and sialylation of IgG antibodies havebeen reported. Parekh et al., Nature 316, 452 (1985), Rademacher et al.,Proc. Natl. Acad. Sci. USA 91, 6123 (1994), Matsumoto et al., 128, 621(2000), Holland et al., Biochim. Biophys. Acta December 27; [Epub aheadof print] 2005. Variations in IgG glycoforms have also been reported tobe associated with aging and upon immunization, although the in vivosignificance of these alterations have not been determined. Shikata etal., Glycoconj. J. 15, 683 (1998), Lastra, et al., Autoimmunity 28, 25(1998).

Accordingly, there is a need for the development of methods for thegeneration of polypeptides that would account for the disparateobservations of IVIG properties in vivo.

SUMMARY OF INVENTION

The present invention fills the foregoing need by providing such methodsand molecules. In one aspect, the invention provides an isolatedpolypeptide containing at least one IgG Fc region, having alteredproperties compared to an unpurified antibody preparation, whereinsialylation of the isolated polypeptide is higher than the sialylationof the unpurified antibody preparation. In one embodiment, the isolatedpolypeptide containing at least one IgG Fc region is glycosylated withat least one galactose moiety connected to a respective terminal sialicacid moiety by a α2,6 linkage, and wherein said polypeptide having ahigher anti-inflammatory activity as compared to an unpurified antibody.In one embodiment the isolated polypeptide containing at least one IgGFc region is glycosylated with at least one galactose moiety connectedto a respective terminal sialic acid moiety by a α2,6 linkage, andwherein said polypeptide having a reduced binding to an Fc activatingreceptor as compared to an unpurified antibody preparation. In a furtherembodiment the Fc activating receptor is selected from the groupconsisting of FcγRIIA, FcγRIIC and FcγRIIIA.

In one aspect, the isolated polypeptide is derived from a recombinantsource.

In another aspect, the instant invention provides a pharmaceuticalformulation comprising a polypeptide containing at least one Fc regionhaving a higher anti-inflammatory activity, in combination with asuitable carrier or diluent.

In another aspect, the invention provides a method of modulatingproperties of a polypeptide comprising an Fc region comprising alteringthe sialylation of the polysaccharide chain of the Fc region.

In one embodiment the method comprises: providing an unpurified sourceof the polypeptide containing at least one Fc region, said unpurifiedsource of the polypeptide containing at least one Fc region comprising aplurality of the polypeptides containing at least one Fc region having apolysaccharide chain comprising a terminal sialic acid connected to agalactose moiety through a α2,6 linkage, and a plurality of thepolypeptides containing at least one Fc region lacking a polysaccharidechain comprising a terminal sialic acid connected to a galactose moietythrough the α2,6 linkage; and increasing the ratio of the plurality ofthe polypeptides containing at least one Fc region having thepolysaccharide chain comprising the terminal sialic acid connected tothe galactose moiety through the α2,6 linkage to the plurality of thepolypeptide containing at least one Fc region lacking the polysaccharidechain comprising the terminal sialic acid connected to the galactosemoiety through the α2,6 linkage.

In yet another embodiment the invention provides a method of treating aninflammatory disease comprising administering to a subject in needthereof a therapeutic composition comprising a plurality of isolatedpolypeptides, each containing at least one IgG Fc region, wherein afirst portion of the respective Fc regions comprises respectivecarbohydrate chains having galactose moieties connected to respectiveterminal sialic acid moieties by 2,6 linkage; a dose of the therapeuticcomposition is smaller than a dose of a second composition whichcomprises a plurality of isolated polypeptides, each containing at leastone IgG Fc region, having a second portion of the respective Fc regionscomprising respective carbohydrate chains having galactose moietiesconnected to respective terminal sialic acid moieties by 2,6 linkage;and either the first portion is greater than the second portion, wherebythe dose of the therapeutic composition and the dose of the secondcomposition suppress inflammation to substantially the same extent, orthe first portion is greater than the second portion, whereby thetherapeutic composition suppresses inflammation to substantially agreater extent than an equal dose of the second composition.

In another aspect, the invention provides a method for controlling theproperties of an Fc-containing molecule, comprising altering thesialylation of the oligosaccharides in the Fc region. In differentembodiments, the sialylation of the Fc region is increased or decreased.

In another aspect, the invention provides methods of treating diseasescomprising administering to a patient in need thereof a therapeuticallyeffective amount of a protein comprising Fc region with alteredoligosaccharide sialylayion. In different embodiments, the sialylationis increased or decreased. The disease may be selected fromoncology-related disorders and diseases or conditions associated withinflammation. In different embodiments, the disease or condition isrheumatoid arthritis or inflammatory bowel disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are footprint histograms of MALDI-TOF analysis of SNA⁺ FClinkages, where the footprint histogram of enriched galactose-sialicacid structures with in vivo anti-inflammatory activity (1A) wascompared to histograms from sialic acid linkage standards, α2-3sialyllactose (1B) and α2-6 sialyllactose (1C).

FIG. 2 summarizes experiments demonstrating that enrichment of α2,6linkages between sialic acid and galactose improves anti-inflammatoryproperties of IVIG Fc fragments.

FIGS. 3A and 3B are a group of photographs (3A) and a diagram (3B)demonstrating that removal of α2,6 linkages between sialic acid andgalactose attenuates anti-inflammatory properties of IVIG Fc fragments.

FIG. 4 demonstrates that reduced cytotoxicity does not depend on thelinkage between galactose and sialic acid.

FIGS. 5A and 5B are a group of photographs (5A) and a diagram (5B)demonstrating that the in vivo anti-inflammatory activity of the 2,6sialylated IgG Fc is solely a property of the IgG Fc glycan.

FIGS. 6A-6D illustrate the hierarchy of antibody-isotype mediatedeffector functions in vivo. Shown are the B16-F10 lung metastasis andplatelet depletion models in C57BL/6 mice (mean±SEM). (A and B) Micewere injected with B16-F10 melanoma cells followed by injection ofTA99-isotype switch variants or control antibodies (200 μg perinjection) on days 0, 2, 4, 7, 9 and 11. Mice were sacrificed 15 daysafter tumor cell injection and the number of surface lung metastasis wasevaluated. Asterisk indicates P<0.0001; double asterisks P<0.01. (C andD) Mice were injected with 4 μg of 6A6-antibody switch variants andplatelet counts were determined at the indicated time points (C). (D)The platelet count 4 hours after injection of the 6A6 isotype variantsis shown as the percentage of the total platelet count before antibodyinjection.

FIGS. 7A and 7B illustrate that IgG2a-mediated effects are independentof the complement cascade in vivo (mean+/−SEM). (A) Mice deficient forcomplement receptor 2 (CR2−/−) or complement component C3 or C4 (C4−/−)were injected with B16-F10 melanoma cells and treated with 100 μg of theTA99-IgG2a antibody per injection. After 15 days animals were sacrificedand lung surface metastasis count was determined. Asterisk indicatesP<0.0001. B) C57BL/6, γ-chain−/−, CR2−/−, and C4−/− mice were injectedwith 4 μg of the 6A6-IgG1, -IgG2a, or IgG2b antibody isotypes andplatelet counts were determined before and hours after antibodyinjection (Nimmerjahn, et al., (2005). Shown is the platelet count 4hours after antibody injection relative to the platelet count beforeinjection percent. The experiments were done twice with 3-4 animals pergroup.

FIGS. 8A-8E illustrate the Fcγ receptor dependence of antibodyisotype-mediated effector functions (A and B). Mice deficient for thecommon γ-chain (γ−/−), or α chains of activation Fcγ-receptor I(FcγRI−/−) or III (FcγRIII−/−) were injected with B16-F10 melanoma cellsand treated with 100 μg of the TA99-IgG2a antibody or PBS (mock) asdescribed. At day 15 after tumor cell injection mice were sacrificed,lungs were prepared (A) and the number of lung surface metastasis wasquantified (B) (mean+/−SEM). Asterisk indicates P<0.0001. The experimentwas performed twice with 5 mice per group. (C and D) FcγRI−/− mice wereinjected with B16-FlO melanoma cells and treated with the TA99-IgG2aantibody at 100 μg per injection. At days 0, 2 and 4 after tumor cellinjections mice were injected with 200 μg of an FcγRIV blocking antibodyor an isotype matched control antibody. Lungs were prepared at day 15after tumor cell injection (C) and lung surface metastasis werequantified (D) (mean+/−SEM). Asterisk indicates P<0.001. (E) Theindicated mouse strains were injected with 4 μg of the 6A6-isotypeswitch variants and the platelet count was determined 4 hours afterinjection of the respective antibody variants (mean+/−SEM). To blockimmune complex binding to FcγRIV mice were injected with 200 μg of anFcγRIV-blocking antibody (Nimmerjahn, F., et al., (2005)). Shown is therelative platelet count 4 hours after antibody injection in percent.Experiments were performed twice with 4-6 mice per group.

FIGS. 9A-9C illustrate differential isotype specific negative regulationby the inhibitory receptor FcγRIIB (A and B). C57BL/6 wild-type orFcγ-receptor IIB deficient (FcγRIIB−/−) mice were injected with B16-F1Omelanoma cells and treated with TA99-IgG1 or IgG2a isotype switchvariants (100 μg per injection). Lungs were prepared on day 15 aftertumor cell injection. The experiment was done twice with 5 mice pergroup; representative lungs are shown in (A) and the quantification in(B). Asterisk indicates P<0.0001; double asterisk indicates P<0.05. (C)C57BL/6 or FcγRIIB−/− mice were injected with 2 μg of the indicated6A6-antibody isotype variants and platelet counts were determined beforeand 4 hours after antibody injection. Shown is the increase in plateletdepletion for the different antibody isotypes in FcγRIIB−/− micecompared to wildtype animals. Shown is one representative out of threeexperiments with 5 mice per group.

FIGS. 10A-10C illustrate that the enhancement of the (A/I) ratio ofmodified antibodies increases their efficacy. (A) Shown is the foldincrease in association constants (K_(A)) for the complement componentC1q and FcγR-receptors I-IV in binding to fucose-containing TA99-IgG1,-IgG2a and -IgG2b isotypes compared to fucose-deficient TA99 isotypeswitch variants. (B and C) C57BL/6 mice were injected with B16-F10melanoma cells and treated with TA99-IgG2b containing fucose orTA99-IgG2b deficient in fucose; Shields, R. L., et al., J Biol Chem 277,26733-40 (2002); Shinkawa, T., et al., J Biol Chem 278, 3466-73. (2003);and Niwa R., et al., Cancer Res 64, 2127-33. (2004)) (50 μg perinjection). Lungs were prepared at day 15 after tumor cell injection.One representative lung out of 4 animals per group (B) and thequantification of the lung surface metastasis count is shown (C).Asterisk indicates P<0.0001.

FIG. 11 illustrates the effect of reducing sialic acid content on the invivo cytotoxicity of an antibody. The effect of sialic acid residues inAsn-297 linked sugar side chains on antibody dependent cytotoxicity invivo is described. Mice (n=4) were injected intravenously with 4 μg ofthe respective 6A6-IgG1 antibodies and platelet counts were determinedbefore and 4 hours after antibody injection. Shown is the plateletdepletion in percent 4 hours after injection of the antibody variants.Abbreviations: SA, sialic acid

FIGS. 12A and 12B illustrate factors that influence Fc-receptordependent activities of antibody isotypes. FIG. 12A indicates individualantibody isotypes have different affinities for activating andinhibitory Fc receptors (see text). Red arrows indicate preferentialinteractions of the indicated antibody isotypes with cellularFc-receptors; black arrows indicate lower affinity interactions. In thecase of IgG2a the broken red arrow indicates that the interaction mightbe blocked as FcγRI is continuously occupied with monomeric IgG2a. Thetable summarizes the actual A/I ratios based on the affinities of theindividual Fc-receptors for the respective antibody isotypes (Nimmerjahnet al., 2005). FIG. 12B indicates the ratio of activating to inhibitoryFc-receptors on immune cells such as DCs, macrophages and neutrophils isregulated by exogenous factors. Cytokines like IL-4, IL-10 or TGF-f3upregulate FcγRIIB thereby setting high thresholds for cell activation,whereas inflammatory mediators downregulate the inhibitory andupregulate the activating Fc-receptors. For therapeutic approachesFcγRIIB mediated inhibition might be circumvented by usingFcγRIIB-blocking antibodies.

DETAILED DESCRIPTION

The inventors have surprisingly found that the cytotoxic andanti-inflammatory response of the IgG Fc domain results from thedifferential sialylation of the Fc-linked core polysaccharide. Thecytotoxicity of IgG antibodies is reduced upon sialylation; conversely,the anti-inflammatory activity of IVIG is enhanced. IgG sialylation isshown to be regulated upon the induction of an antigen-specific immuneresponse, thus providing a novel means of switching IgG from an innate,anti-inflammatory molecule in the steady-state, to a adaptive,pro-inflammatory species upon antigenic challenge. The Fc-sialylatedIgGs bind to a unique receptor on macrophages that in turn upregulatesan inhibitory Fcγ receptor (FcγR) thereby protecting againstautoantibody-mediated pathology. See, generally, Ravetch and Nimmerjahn,J. Experim. Medicine 24(1): 11-15 (2007). The inventors have furthersurprisingly discovered that the anti-inflammatory response depends onthe nature of the linkage between galactose and sialic acid moieties.The observation that the anti-inflammatory activity of IVIG is dependenton a precise glycan structure on the Fc further supports the model thatthe inventors have previously advanced (Y. Kaneko, F. Nimmerjahn, J. V.Ravetch, Science 313, 670 (2006); F. Nimmerjahn, J. V. Ravetch, J ExpMed 204, 11 (2007)) that a specific lectin receptor, and not a canonicalFc receptor, is involved in this pathway. The data underlying thisinvention support a model in which binding of the 2,6 sialylated Fc toits cognate lectin receptor expressed on a population of regulatorymyeloid cells results in the trans upregulation of the inhibitory IgG Fcon effector macrophages, located at sites of inflammation, such as theinflamed joint, thus raising the threshold required for cytotoxic IgGsto engage activation FcRs and trigger inflammatory responses (F.Nimmerjahn, J. V. Ravetch, Science 310, 1510 (2005)).

Accordingly, the instant disclosure provides an advantageous strategy ofcreating and selecting IgGs with desired cytotoxic and anti-inflammatorypotential.

DEFINITIONS

Throughout the present specification and claims, the numbering of theresidues in an immunoglobulin heavy chain is that of the EU index as inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991), which is expressly incorporated herein by reference. The “EUindex as in Kabat” refers to the residue numbering of the human IgG1 EUantibody.

The term “native” or “parent” refers to an unmodified polypeptidecomprising an Fc amino acid sequence. The parent polypeptide maycomprise a native sequence Fc region or an Fc region with pre-existingamino acid sequence modifications (such as additions, deletions and/orsubstitutions).

The term “polypeptide” refers to any fragment of a protein containing atleast one IgG Fc region and fragments thereof, including, withoutlimitation, fully functional proteins, such as, for example, antibodies,e.g., IgG antibodies. When a polypeptide of the invention is compared toan unpurified antibody preparation, such a preparation is typically ablood sample, serum sample, and/or IVIG sample, derived from a mammal,e.g., a human donor. The preparation may be unfractionated or partiallyfractionated but typically comprises only about 2-4% sialylated Fccontaining proteins. Compositions of the invention enriched orformulated to have immunosuppressive activity typically comprise atleast about 5% sialylated Fc containing proteins or more (e.g., 5-10%,10-30%, 30-50%, 50-100% or ranges or intervals thereof).

The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain. The “Fc region” may be a native sequence Fcregion or a variant Fc region. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof.

The “CH2 domain” of a human IgG Fc region (also referred to as “Cγ2”domain) usually extends from about amino acid 231 to about amino acid340. The CH2 domain is unique in that it is not closely paired withanother domain. Rather, two N-linked branched carbohydrate chains areinterposed between the two CH2 domains of an intact native IgG molecule.It has been speculated that the carbohydrate may provide a substitutefor the domain-domain pairing and help stabilize the CH2 domain (Burton,Mol Immunol, 22, 161-206 (1985), which is incorporated herein byreference).

The “CH3 domain” comprises the stretch of residues C-terminal to a CH2domain in an Fc region (i.e., from about amino acid residue 341 to aboutamino acid residue 447 of an IgG).

The term “hinge region” is generally defined as stretching from Glu216to Pro230 of human IgG1 (Burton (1985). Hinge regions of other IgGisotypes may be aligned with the IgG1 sequence by placing the first andlast cysteine residues forming inter-heavy chain S—S bonds in the samepositions.

The term “binding domain” refers to the region of a polypeptide thatbinds to another molecule. In the case of an FcR, the binding domain cancomprise a portion of a polypeptide chain thereof (e.g., the α chainthereof) which is responsible for binding an Fc region. One exemplarybinding domain is the extracellular domain of an FcR chain.

A “functional Fc region” possesses at least a partial “effectorfunction” of a native sequence Fc region. Exemplary “effector functions”include C1q binding; complement dependent cytotoxicity; Fc receptorbinding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; down regulation of cell surface receptors (e.g., B cellreceptor; BCR), etc. Such effector functions generally require the Fcregion to be combined with a binding domain (e.g., an antibody variabledomain) and can be assessed using various assays as herein disclosed,for example. The term also includes Fc fragments provided the fragmentcontains at least one amino acid residue that is glycosylated orsuitable for glycosylation as described herein.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” as appreciated by one of ordinary skill in the art comprisesan amino acid sequence which differs from that of a native sequence Fcregion by virtue of at least one “amino acid modification.” Preferably,the variant Fc region has at least one amino acid substitution comparedto a native sequence Fc region or to the Fc region of a parentpolypeptide, e.g., from about one to about ten amino acid substitutions,and preferably from about one to about five amino acid substitutions ina native sequence Fc region or in the Fc region of the parentpolypeptide. The variant Fc region herein will preferably possess atleast about 80% homology with a native sequence Fc region and/or with anFc region of a parent polypeptide, and more preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith, even more preferably, at least about 99% homology therewith.

The term “altered glycosylation” refers to a polypeptide, as definedabove, be it native or modified, in which the carbohydrate addition tothe heavy chain constant region is manipulated to either increase ordecrease specific sugar components. For example, polypeptides, such as,for example, antibodies, prepared in specific cell lines, such as, forexample, Lec2 or Lec3, may be deficient in the attachment of sugarmoieties such as fucose and sialic acid.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In one embodiment of theinvention, FcR is a native sequence human FcR. In another embodiment,FcR, including human FcR, binds an IgG antibody (a gamma receptor) andincludes receptors of the FcγRI, FcγRII, and FcγRIII subclasses,including allelic variants and alternatively spliced forms of thesereceptors. FcγRII receptors include FcγRIIA (an “activating receptor”)and FcγRIIB (an “inhibiting receptor”), which have similar amino acidsequences that differ primarily in the cytoplasmic domains thereof.Activating receptor FcγRIIA contains an immunoreceptor tyrosine-basedactivation motif (ITAM) in its cytoplasmic domain. Inhibiting receptorFcγRIIB contains an immunoreceptor tyrosine-based inhibition motif(ITIM) in its cytoplasmic domain (see review in Daron, Annu Rev Immunol,15, 203-234 (1997); FcRs are reviewed in Ravetch and Kinet, Annu RevImmunol, 9, 457-92 (1991); Capel et al., Immunomethods, 4, 25-34 (1994);and de Haas et al., J Lab Clin Med, 126, 330-41 (1995), Nimmerjahn andRavetch 2006, Ravetch Fc Receptors in Fundamental Immunology, ed WilliamPaul 5^(th) Ed. each of which is incorporated herein by reference).

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to anin vitro or in vivo cell-mediated reaction in which cytotoxic cells thatexpress FcRs (e.g., monocytic cells such as natural killer (NK) cellsand macrophages) recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. In principle, any effectorcell with an activating FcγR can be triggered to mediate ADCC. One suchcell, the NK cell, expresses FcγRIII only, whereas monocytes, dependingon their state of activation, localization, or differentiation, canexpress FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoieticcells is summarized in Ravetch and Bolland, Annu Rev Immunol, (2001),which is incorporated herein by reference.

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at least onetype of an activating Fc receptor, such as, for example, FcγRIII andperform ADCC effector function. Examples of human leukocytes whichmediate ADCC include peripheral blood mononuclear cells (PBMC), naturalkiller (NK) cells, monocytes, and neutrophils, with PBMCs and NK cellsbeing preferred. The effector cells may be isolated from a native sourcethereof, e.g., from blood or PBMCs as described herein.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity.

The phrase “sialic acid content” of an antibody refers both to the totalnumber of sialic acid residues on an Fc region of a heavy chain of anantibody and to the ratio of sialylated antibodies to asialylatedantibodies in an unpurified antibody preparation, unless the phrase isin a context clearly suggesting that another meaning is intended. Asmentioned above in the BACKGROUND section, IgG contains a single,N-linked glycan at Asn²⁹⁷ in the CH2 domain on each of its two heavychains. The N-linked glycan structure can end with no galactose, onegalactose, or two galactoses, referred as G0, G1, or G2. In a sialylatedantibody, a sialic acid (S) is linked to a galactose (G) with theformation of an α-linkage between the two saccharides. Once bothgalactoses are linked to sialic acids, the sialylated antibody has aglycoform with 2 galactoses (G2) linked with 2 sialic acids (S2), i.e.,a G2S2 sialylated glycoform.

“Antibody fragments”, as defined for the purpose of the presentinvention, comprise a portion of an intact antibody, generally includingthe antigen binding or variable region of the intact antibody or the Fcregion of an antibody which retains FcR binding capability. Examples ofantibody fragments include linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.The antibody fragments preferably retain at least part of the hinge andoptionally the CH1 region of an IgG heavy chain. More preferably, theantibody fragments retain the entire constant region of an IgG heavychain, and include an IgG light chain.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,Nature, 256, 495-497 (1975), which is incorporated herein by reference,or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567, which is incorporated herein by reference). The monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in Clackson et al., Nature, 352, 624-628 (1991) andMarks et al., J Mol Biol, 222, 581-597 (1991), for example, each ofwhich is incorporated herein by reference.

In other embodiments of the invention, the polypeptide containing atleast one IgG Fc region may be fused with other protein fragments,including, without limitation, whole proteins. A person of ordinaryskill in the art will undoubtedly appreciate that many proteins may befused with the polypeptide of the present invention, including, withoutlimitation, other immunoglobulins, especially, immunoglobulins lackingtheir respective Fc regions. Alternatively, other biologically activeproteins or fragments thereof may be fused with the polypeptide of thepresent invention, as described, for example, in the U.S. Pat. No.6,660,843, which is incorporated herein by reference. This embodiment isespecially advantageous for delivery of such biologically activeproteins or fragments thereof to cells expressing Fc receptors. Further,different markers, such as, for example, GST tag or green fluorescentprotein, or GFP, may be used.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (see U.S. Pat. No.4,816,567; Morrison et al., Proc Natl Acad Sci USA, 81, 6851-6855(1984); Neuberger et al., Nature, 312, 604-608 (1984); Takeda et al.,Nature, 314, 452-454 (1985); International Patent Application No.PCT/GB85/00392, each of which is incorporated herein by reference).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR residues are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature, 321,522-525 (1986); Riechmann et al., Nature, 332, 323-329 (1988); Presta,Curr Op Struct Biol, 2, 593-596 (1992); U.S. Pat. No. 5,225,539, each ofwhich is incorporated herein by reference.

The polypeptides of the instant invention may be recombinantly produced,for example, from a cDNA, such as, for example SEQ ID NO: 1. Thepolypeptides of different embodiments include Fc regions or functionalfragments thereof.

The polypeptides containing at least one IgG Fc region include those inwhich specific amino acid substitutions, additions or deletions areintroduced into a parental sequence through the use of recombinant DNAtechniques to modify the genes encoding the heavy chain constant region.The introduction of these modifications follows well-establishedtechniques of molecular biology, as described in manuals such asMolecular Cloning (Sambrook and Russel, (2001)). In addition, thepolypeptides with at least one Fc region will include those polypeptideswhich have been selected to contain specific carbohydrate modifications,obtained either by expression in cell lines known for theirglycosylation specificity (Stanley P., et al., Glycobiology, 6, 695-9(1996); Weikert S., et al., Nature Biotechnology, 17, 1116-1121 (1999);Andresen D C and Krummen L., Current Opinion in Biotechnology, 13,117-123 (2002)) or by enrichment or depletion on specific lectins or byenzymatic treatment (Hirabayashi et al., J Chromatogr B Analyt TechnolBiomed Life Sci, 771, 67-87 (2002); Robertson and Kennedy,Bioseparation, 6, 1-15 (1996)). It is known in the art that quality andextent of antibody glycosylation will differ depending on the cell typeand culture condition employed. (For example, Patel et al., Biochem J,285, 839-845 (1992)) have reported that the content of sialic acid inantibody linked sugar side chains differs significantly if antibodieswere produced as ascites or in serum-free or serum containing culturemedia. Moreover, Kunkel et al., Biotechnol Prog, 16, 462-470 (2000) haveshown that the use of different bioreactors for cell growth and theamount of dissolved oxygen in the medium influenced the amount ofgalactose and sialic acid in antibody linked sugar moieties. Thesestudies, however, did not address how varying levels of sialic acidresidues influence antibody activity in vivo.

Host Expression Systems

The polypeptide of the present invention can be expressed in a hostexpression systems, i.e., host cells, capable of N-linked glycosylation.Typically, such host expression systems may comprise bacterial, fungal,plant, vertebrate or invertebrate expression systems. In one embodimentthe host cell is a mammalian cell, such as a Chinese hamster ovary (CHO)cell line, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cell line (COS)(e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cell (e.g.NS/0), Baby Hamster Kidney (BHK) cell line (e.g. ATCC CRL-1632 or ATCCCCL-10), or human cell (e.g. HEK 293 (ATCC CRL-1573) or 293T (ATCCCRL-11268)), or any other suitable cell line, e.g., available frompublic depositories such as the American Type Culture Collection,Rockville, Md. Further, an insect cell line, such as a Lepidoptora cellline, e.g. Sf9, a plant cell line, a fungal cell line, e.g., yeast suchas, for example, Saccharomyces cerevisiae, Pichia pastoris, Hansenulaspp., or a bacterial expression system based on Bacillus, such as B.subtilis, or Eschericiae coli can be used. It will be appreciated by oneof ordinary skill in the art that in some cases modifications to hostcells may be required to insure that N-linked glycosylation and glycanmaturation occur to result in a complex, biantennary sugar as typicallyfound on the Fc domain of human IgG.

Therapeutic Formulations

Therapeutic formulations comprising the polypeptides containing at leastone IgG Fc region can be prepared for storage by mixing the polypeptidesof the present invention having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers(see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenyl, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in a microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

In preferred embodiments, the formulations to be used for in vivoadministration are sterile. The formulations of the instant inventioncan be easily sterilized, for example, by filtration through sterilefiltration membranes.

Sustained-release preparations may also be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the modified antibody, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (see, e.g., U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Creation of Sialylated Polypeptides Containing at Least One IgG FcRegion.

The polypeptides of the present invention can be further purified ormodified so that they have an increased amount of sialic acid comparedto unmodified and/or unpurified antibodies. Multiple methods exist toreach this objective. In one method, the source of unpurifiedpolypeptides, such as, for example, IVIG, is passed through the columnhaving lectin, which is known to bind sialic acid. A person of theordinary skill in the art will appreciate that different lectins displaydifferent affinities for α2,6 versus α2,3 linkages between galactose andsialic acid. Thus, selecting a specific lectin will allow enrichment ofantibodies with the desired type of linkage between the sialic acid andthe galactose. In one embodiment, the lectin is isolated from Sambuccusnigra. A person of the ordinary skill in the art will appreciate thatthe Sambuccus nigra agglutinin (SNA) is specific for sialic acids linkedto galactose or N-acetylgalactosamine by α(2-6) linkages. Shibuya et al,J. Biol. Chem., 262: 1596-1601 (1987). In contrast, the Maakia amurensis(“MAA”) lectin binds to sialic acid linked to galactose by α(2-3)linkages. Wang et al, J Biol Chem., 263: 4576-4585 (1988).

Thus, a fraction of the polypeptides containing at least one IgG Fcregion having a desired linkage between the galactose and the sialicacid will be retained in the column while a fraction lacking suchlinkage will pass through. The sialylated fraction of the polypeptidescontaining at least one IgG Fc region can be eluted by another wash witha different stringency conditions. Thus, it is possible to obtain apreparation of the polypeptide of the present invention wherein thecontent of sialic acid is increased compared to the normal content.Further, one may employ an enzymatic reaction with a sialyltransferaseand a donor of sialic acid as described, for example, in the U.S. Pat.No. 20060030521.

Suitable non-limiting examples of sialyltransferase enzymes useful inthe claimed methods are ST3Gal III, which is also referred to asα-(2,3)sialyltransferase (EC 2.4.99.6), and α-(2,6)sialyltransferase (EC2.4.99.1).

Alpha-(2,3)sialyltransferase catalyzes the transfer of sialic acid tothe Gal of a Gal-β-1,3GlcNAc or Gal-β-1,4GlcNAc glycoside (see, e.g.,Wen et al., J. Biol. Chem. 267: 21011 (1992); Van den Eijnden et al., J.Biol. Chem. 256: 3159 (1991)) and is responsible for sialylation ofasparagine-linked oligosaccharides in glycopeptides. The sialic acid islinked to a Gal with the formation of an α-linkage between the twosaccharides. Bonding (linkage) between the saccharides is between the2-position of NeuAc and the 3-position of Gal. This particular enzymecan be isolated from rat liver (Weinstein et al., J. Biol. Chem. 257:13845 (1982)); the human cDNA (Sasaki et al. (1993) J. Biol. Chem. 268:22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem. 269: 1394-1401)and genomic (Kitagawa et al. (1996) J. Biol. Chem. 271: 931-938) DNAsequences are known, facilitating production of this enzyme byrecombinant expression.

Activity of α-(2,6)sialyltransferase results in 6-sialylatedoligosaccharides, including 6-sialylated galactose. The name“α-(2,6)sialyltransferase” refers to the family of sialyltransferasesattaching sialic acid to the sixth atom of the acceptor polysaccharide.Different forms of a-(2,6)sialyltransferase can be isolated fromdifferent tissues. For example, one specific form of this enzyme, ST6GalII, can be isolated from brain and fetal tissues. Krzewinski-Recchi etal., Eur. J. Biochem. 270, 950 (2003).

In addition, a person of average skill in the art will appreciate thatcell culture conditions can be manipulated to change the sialylationrate. For example, to increase the sialic acid content, production rateis decreased and osmolality is generally maintained within a lowermargin suitable for the particular host cell being cultured. Osmolalityin the range from about 250 mOsm to about 450 mOsm is appropriate forincreased sialic acid content. This and other suitable cell cultureconditions are described in, e.g., U.S. Pat. No. 6,656,466. Patel etal., Biochem J, 285, 839-845 (1992) have reported that the content ofsialic acid in antibody linked sugar side chains differs significantlyif antibodies were produced as ascites or in serum-free or serumcontaining culture media. Moreover, Kunkel et al., Biotechnol. Prog.,16, 462-470 (2000) have shown that the use of different bioreactors forcell growth and the amount of dissolved oxygen in the medium influencedthe amount of galactose and sialic acid in antibody linked sugarmoieties.

In another embodiment, host cells, such as, for example, immortalizedhuman embryonic retina cells, may be modified by introducing a nucleicacid encoding a sialyltransferase such as, for example, anα-2,3-sialyltransferase or an α-2,6-sialyltransferase, operably linkedto a promoter, such as, for example, a CMV promoter. Theα-2,3-sialyltransferase may be the human α-2,3-sialyltransferase, knownas SIAT4C or STZ (GenBank accession number L23767), and described, forexample, in the U.S. Pat. No. 20050181359.

The nucleic acid encoding the sialyltransferase may be introduced intothe host cell by any method known to a person of ordinary skill in theart. Suitable methods of introducing exogenous nucleic acid sequencesare also described in Sambrook and Russel, Molecular Cloning: ALaboratory Manual (3^(rd) Edition), Cold Spring Harbor Press, N Y, 2000.These methods include, without limitation, physical transfer techniques,such as, for example, microinjection or electroporation; transfections,such as, for example, calcium phosphate transfections; membrane fusiontransfer, using, for example, liposomes; and viral transfer, such as,for example, the transfer using DNA or retroviral vectors.

The polypeptide containing at least one IgG Fc region may be recoveredfrom the culture supernatant and can be subjected to one or morepurification steps, such as, for example, ion-exchange or affinitychromatography, if desired. Suitable methods of purification will beapparent to a person of ordinary skill in the art.

A person of ordinary skill in the art will appreciate that differentcombinations of sialylation methods, disclosed above, can lead toproduction of the polypeptides containing at least one IgG Fc regionwith an extremely high level of sialylation. For example, one canexpress the polypeptide containing at least one IgG Fc region in thehost cells overexpressing sialyltransferase, as described above, andthen further enrich the sialylated fraction of these polypeptides by,for example, sialylating these polypeptides in an enzymatic reactionfollowed by an affinity chromatography using lectin-containing columns.Similarly, an enzymatic reaction followed by affinity chromatography maybe used for IVIG source of the polypeptides containing at least one IgGFc region.

To examine the extent of glycosylation on the polypeptides containing atleast one IgG Fc region, these polypeptides can be purified and analyzedin SDS-PAGE under reducing conditions. The glycosylzation can bedetermined by reacting the isolated polypeptides with specific lectins,or, alternatively as would be appreciated by one of ordinary skill inthe art, one can use HPLC followed by mass spectrometry to identify theglycoforms. (Wormald, M R et al., Biochem 36:1370 (1997).

To describe the instant invention in more details, several non-limitingillustrative examples are given below.

EXAMPLES Example 1 IVIG with Increased Sialic Acid Content ExhibitsDecreased Cytotoxicity

To determine if specific glycoforms of IgG are involved in modulatingthe effector functions of antibodies the role of specific, Asn²⁹⁷-linkedcarbohydrates in mediating the cytotoxicity of defined IgG monoclonalantibodies was explored. The anti-platelet antibodies, derived from the6A6 hybridoma, expressed as either an IgG1, 2a or 2b switch variant in293 cells as previously described (6), were analyzed by massspectroscopy to determine their specific carbohydrate composition andstructure. These antibodies contain minimal sialic acid residues.Enrichment of the sialic acid containing species by Sambucus nigralectin affinity chromatography yielded antibodies enriched 60-80 fold insialic acid content. Comparison of the ability of sialylated andasialylated 6A6-IgG1 and 2b antibodies to mediate platelet clearancerevealed an inverse correlation between sialylation and in vivoactivity. Sialylation of 6A6 IgG antibodies resulted in a 40-80%reduction in biological activity.

To determine the mechanism of this reduction in activity surface plasmonresonance binding was performed on these antibodies for each of themouse FcYRs and to its cognate antigen.

Surface plasmon resonance analysis was performed as described inNimmerjahn and Ravetch, Science 310, 1510 (2005). Briefly, 6A6 antibodyvariants containing high or low levels of sialic acid residues in theirsugar side chains were immobilized on the surface of CM5 sensor chips.Soluble Fcγ-receptors were injected at different concentrations throughflow cells at room temperature in HBS-EP running buffer (10 mM Hepes, pH7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20) at a flow rateof 30 uI/min. Soluble Fc-receptors were injected for 3 minutes anddissociation of bound molecules was observed for 7 minutes. Backgroundbinding to control flow cells was subtracted automatically. Controlexperiments were performed to exclude mass transport limitations.Affinity constants were derived from sensorgram data using simultaneousfitting to the association and dissociation phases and global fitting toall curves in the set. A 1:1 Langmuir binding model closely fitted theobserved sensorgram data and was used in all experiments.

A 5-10 fold reduction in binding affinity was observed for thesialylated forms of these antibodies to their respective activatingFcyRs as compared to their asialylated counterparts, while nodifferences in binding affinity for the antigen were observed. SinceIgG2b binds with a higher affinity to its activation receptor, FcyRIV,when compared to IgG1 binding to its activation receptor FcyRIII, theeffect of sialylation was to generate a binding affinity for IgG2b forits activation receptor FcyRIV that was comparable to that ofasialylated IgG1 binding to its activation receptor FcYRlII. This effectof this quantitative difference in activation receptor binding resultedin sialylated IgG2b displaying an in vivo activity comparable to that ofasialylated IgG1. Similarly, sialylation of IgG1 reduces its already lowbinding affinity for its activation receptor FcyRlll by a factor of 7thereby generating a physiologically inactive antibody. Thus,sialylation of the Asn²⁹⁷ linked glycan structure of IgG resulted inreduced binding affinities to the subclass-restricted activation FcyRsand thus reduced their in vivo cytotoxicity.

To determine the generality of the observation that sialylation of theN-linked glycan of IgG was involved in modulating its in vivoinflammatory activity, we next examined the role of N-linked glycans onthe anti-inflammatory activity of IVIG. This purified IgG fractionobtained from the pooled serum of 5-10,000 donors, when administeredintravenously at high doses (1-2 g/kg), is a widely used therapeutic forthe treatment of inflammatory diseases. Dwyer, N. Engl. J. Med. 326, 107(1992). This anti-inflammatory activity is a property of the Fc fragmentand is protective in murine models of ITP, RA and nephrotoxic nephritis.Imbach et al., Lancet 1, 1228 (1981), Samuelsson et al., Science 291,484 (2001), Bruhns et al., Immunity 18, 573 (2003), Kaneko et al., J.Exp. Med. 203(3):789-97 (2006).

A common mechanism for this anti-inflammatory activity was proposedinvolving the induction of surface expression of the inhibitory FcyRIIBmolecule on effector macrophages, thereby raising the threshold requiredfor cytotoxic IgG antibodies or immune complexes to induce effector cellresponses by activation FcyR triggering. Nimmerjahn and Ravetch,Immunity 24, 19 (2006).

Example 2 Asialylation of IVIG Decreases the Anti-Inflammatory Effect ofIVIG in Mouse Arthritis Model

Mice

C57BL/6 and NOD mice were purchased from the Jackson Laboratory (BarHarbor, Me.). FcγRIIB^(−/−) mice were generated in the inventors'laboratory and backcrossed for 12 generations to the C57BL/6 background.KRN TCR transgenic mice on a C57BU6 background (K/B) were gifts from D.Mathis and C. Benoist (Harvard Medical School, Boston, Mass.) and werebred to NOD mice to generate K/B×N mice. Female mice at 6-10 weeks ofage were used for all experiments and maintained at the RockefellerUniversity animal facility.

Serum was prepared as described previously (Bruhns, et al., Immunity 18,573 (2003)). Briefly, serum is separated from blood collected from theK/B×N mice (6-12 weeks old). Several weeks of serum collection werepooled together and frozen in aliquots to be used in all the experimentsdescribed here. One intravenous injection of 1.5× diluted K/B×N serum (4μl of pooled K/B×N serum per gram of mouse) induced arthritis. Arthritiswas scored by clinical examination. Indices of all four paws are added:0 [unaffected], 1 [swelling of one joint], 2 [swelling of more than onejoint], and 3 [severe swelling of the entire paw]. IVIG is injected 1 hrbefore K/B×N serum injection. Some mice received 5 μg of plateletdepleting 6A6-IgG2b antibody, and platelet counts were determined at 0,4, and 24 hours post treatment using an Advia 120 haematology system(Bayer). All experiments were done in compliance with federal laws andinstitutional guidelines and have been approved by the RockefellerUniversity (New York, N.Y.).

Antibodies and Soluble Fc Receptors

6A6 antibody switch variants were produced by transient transfection of293T cells followed by purification via protein G as described.Nimmerjahn and Ravetch, Science 310, 1510 (2005). Sialic acid richantibody variants were isolated from these antibody preparations bylectin affinity chromatography with Sambucus nigra agglutinin (SNA)agarose (Vector Laboratories, Burlingame, Calif.). Enrichment for sialicacid content was verified by lectin blotting (see below). Humanintravenous immune globulin (IVIG, 5% in 10% maltose, chromatographypurified) was purchased from Octapharma (Hemdon, Va.). Digestion ofhuman IVIG was performed as described. Kaneko Y. et al., Exp. Med.203(3):789-97 (2006). Briefly, IVIG was digested by 0.5 mg/ml papain for1 hr at 37° C., and stopped by the addition of 2.5 mg/ml iodoasetamide.Fab and Fc resulting fragments were separated from non-digested IVIG ona HiPrep 26/60 S-200HR column (GE Healthcare, Piscataway, N.J.),followed by purification of Fc and Fab fragments with a Protein G column(GE Healthcare) and a Protein L column (Pierce, Rockford, Ill.).Fragment purity was checked by immunoblotting using anti-human IgG Fabor Fc-specific antibodies. (Jackson ImmunoResearch, West Grove, Pa.).Purity was judged to be greater than 99%. The F4/80 antibody was fromSerotec (Oxford, UK). The Ly 17.2 antibody was from Caltag (Burlingame,Calif.). Sheep anti-glomerular basement membrane (GBM) antiserum(nephrotoxic serum, NTS) was a gift from M. P. Madaio (University ofPennsylvania, Philadelphia, Pa.). Soluble Fc receptors containing aC-terminal hexa-hisitidine tag were generated by transient transfectionof 293T cells and purified from cell culture supernatants with Ni-NTAagarose as suggested by the manufacturer (Qiagen).

IVIG was treated with neuraminidase and the composition and structure ofthe resulting preparation was analyzed by mass spectroscopy. Nodetectable sialic acid containing glycans remained after neuraminidasetreatment. These IgG preparations were then tested for their ability toprotect mice from joint inflammation induced by passive transfer of K×Nserum, an IgG 1 immune complex-mediated inflammatory disease model.De-sialylation with neuraminidase abrogated the protective effect of theIVIG preparation in the K×N serum induced arthritis model. This loss ofactivity was not the result of reduced serum half-life of theasialylated IgG preparations or the result of changes to the monomericcomposition or structural integrity of the IgG. Removal of all glycanswith PNGase had a similar effect and abrogated the protective effect ofIVIG in vivo.

Example 3 IVIG Fraction with Enriched Sialic Acid Content DecreasesInflammation in Mouse Arthritis Model

Preparation of IVIG with an Increased Content of Sialic Acid

Since sialic acid appeared to be required for the anti-inflammatoryactivity of IVIG, the basis for the high dose requirement (1 g/kg) forthis anti-inflammatory activity could be the limiting concentration ofsialylated IgG in the total IVIG preparation. The IVIG was fractionatedon an SNA-lectin affinity column to obtain IgG molecules enriched forsialic acid modified glycan structures.

These sialic acid enriched fractions were tested for protective effectsin the K×N serum transfer arthritis model as compared to unfractionatedIVIG. A 10 fold enhancement in protection was observed for theSNA-binding fraction, such that equivalent protection was obtained at0.1 g/kg of SNA-enriched IVIG as compared to 1 g/kg of unfractionatedIVIG. The serum half-life and IgG subclass distribution of the SNAenriched fraction was equivalent to that of unfractionated IVIG. Theeffect of sialylation was specific to IgG; sialylated N-linkedglycoproteins such as fetuin or transferrin with similar biantennary,complex carbohydrate structures had no statistically significantanti-inflammatory activity at equivalent molar concentrations of IgG.Finally, the mechanism of protection of the sialylated IVIG preparationwas similar to unfractionated IVIG in that it was dependent on FcγRIIBexpression and resulted in the increased expression of this inhibitoryreceptor on effector macrophages.

Example 4 The Increased Anti-Inflammatory Response of IVIG withIncreased Sialic Acid Content is Mediated by Sialylation of the N-LinkedGlycan on the Fc Domain

Since the polyclonal IgG in IVIG may also contain O and N linked glycanson the light chains or heavy chain variable domains that can besialylated, we confirmed that the increase in anti-inflammatory activityof the SNA-enriched IgG preparation resulted from increased sialylationof the N-linked glycosylation site on the Fc. Fc fragments weregenerated from unfractionated and SNA fractionated IVIG and tested fortheir in vivo activity. As observed for intact IgG, SNA-purified Fcfragments were enhanced for their protective effect in vivo whencompared to Fc fragments generated from unfractionated IVIG. Incontrast, Fab fragments displayed no anti-inflammatory activity in thisin vivo assay. Thus, the high dose requirement for the anti-inflammatoryactivity of IVIG can be attributed to the minor contributions ofsialylated IgG present in the total preparation. Enrichment of thesefractions by sialic acid binding lectin chromatography consequentlyincreased the anti-inflammatory activity.

These results using passive immunization of IgG antibodies indicatedthat the ability of IgG to switch from a pro-inflammatory to ananti-inflammatory species is influenced by the degree of sialylation ofthe N-linked glycan on the Fc domain.

Example 5 Increase of Anti-Inflammatory Activity, Mediated bySialylation of IgG, Occurs During an Active Immune Response

Murine Model for Goodpasture's Disease

In this model, mice are first sensitized with sheep IgG together withadjuvant and four days later injected with a sheep anti-mouse glomerularbasement membrane preparation (nephrotoxic serum, NTS). Briefly, micewere pre-immunized intraperitoneally with 200 μg of sheep IgG (SEROTEC)in CFA, followed by intravenous injection of 2.5 μl of NTS serum pergram of body weight four days later. Blood was collected fromnon-treated control mice four days after the anti-GBM anti-seruminjection, and serum IgG was purified by Protein G (GE Healthcare,Princeton, N.J.) and SEPHAROSE-bound sheep IgG column, generated bycovalently coupling sheep IgG on NHS-activated SEPHAROSE-column (GEHealthcare, Princeton, N.J.), affinity chromatography.

Pre-sensitization followed by treatment with NTS induces mouse IgG2banti-sheep IgG antibodies (NTN immunized). Kaneko Y. et al., Exp. Med.,203:789 (2006). Mouse IgG2b antibodies are deposited in the glomerulustogether with the NTS antibodies and result in an acute and fulminantinflammatory response by the IgG2b mediated activation of FcyRIV oninfiltrating macrophages. In the absence of pre-sensitizationinflammation is not observed, indicating that the mouse IgG2b anti-sheepIgG antibodies are the mediators of the inflammatory response.

To determine if active immunization resulting in pro-inflammatory IgG isassociated with a change in sialylation, serum IgG and IgM frompreimmune and NTS immunized mice were characterized for sialic acidcontent by SNA lectin binding. Total IgG sialylation was reduced onaverage by 40% in immunized mice as compared to the unimmunizedcontrols. The effect was specific for IgG; sialylation of IgM wasequivalent pre and post immunization. This difference in sialylation wasmore pronounced when the sheep specific IgG fraction from mouse serumwas analyzed, showing a 50-60% reduction in sialylation compared topreimmune IgG.

These results were confirmed by MALDI-TOF-MS analysis. Monosaccharidecomposition analysis was performed by UCSD Glycotechnology Core Resource(San Diego, Calif.). Glycoprotein samples were denatured with SDS and2-mercaptoethanol, and digested with PNGase F. The released mixedN-glycans were purified by reversed-phase HPLC and solid-phaseextraction, and then exposed hydroxyl groups of the N-glycans weremethylated. The resulting derivatized saccharides were purified again byreversed-phase HPLC and subject to MALDI-TOF-MS.

The analysis of the pre and post immunization IgGs confirmed that thechanges in the N-glycan structure were specific to the terminal sialicacids moieties. The mouse IgG2b anti-sheep antibodies that weredeposited in the glomeruli, previously shown to be responsible forengagement of the FcyRIV bearing, infiltrating macrophages displayedreduced sialic acid content as compared to the pre-immunized controls.

Example 6 Analysis of Linkages Between Sialic Acid and Galactose in IVIG

Sequential Maldi-Tof analysis of SNA⁺ (Sambuccus Nigra Agglutinin) IVIGFc linkages was performed to determine the structure of the sialylatedIgG Fc fraction that was protective in the ITP, RA and nephrotoxicnephritis models described above. Glycan peaks generated in Maldi-TOFwere isolated, further fractionated, and reanalyzed untilgalactose-sialic acid structures were obtained. The footprint histogramof the enriched galactose-sialic acid structures with in vivoanti-inflammatory activity (FIG. 1A) were compared to histograms fromsialic acid linkage standards, α2-3 sialyllactose (FIG. 1B) and α2-6sialyllactose (FIG. 1C). The signature peaks of the standards areidentified by arrows, shown by arrows for α2-3 (FIG. 1B) or α2-6 (FIGS.1A and 1C), respectively, and compared to the peaks obtained from thesample.

Example 7 Enrichment of IVIG Fc Fragments in α2,6 Linkages by In VitroGlycosylation Improves Anti-Inflammatory Properties of IVIG

As shown in FIG. 2A, glycan Maldi-Tof MS analysis of IVIG Fc fragmentsshowed structures ending in no galactose (peak G0), one galactose (peakG1), two galactose (peak G2), or in sialic acid (indicated by a bracketentitled “Terminal sialic acid”). To determine the in vivo activity of2,3 or 2,6 sialylated IgG Fc, samples were treated with sialidase,followed by galactose transferase to convert the G0 (no galactose) andG1 (single_galactose) to G2 (fully galactosylated) to increase potentialsialylation sites. As shown in FIG. 2B hypergalactosylation was verifiedby comparing relative band intensity ratios of terminal galactose asmeasured by ECL and coomassie loading controls. In vitro sialylation wasperformed (FIG. 2C) using either α2-6 sialyltransferase (“ST6Gal”) orα2-3 sialyltransferase (“ST3Gal”) and confirmed by lectin blotting forα2-6 linkages with SNA (top) or α2-3 linkages with ECL (middle) andcoomassie (bottom). To evaluate the ability of in vitro sialylated Fc toinhibit inflammation (FIG. 2D) mice received either 0.66 mg of α2-6sialylated Fcs (black triangles) or 0.66 mg α2-3 sialylated Fcs (redtriangles). 1 hour later, 0.2 ml of K/B×N sera was administered, and theswelling of footpads (clinical score) was monitored over the next sevendays. Anti-inflammatory activity was observed for the 2,6 sialylated IgGFc fragments but not for the 2,3 sialylated molecules. These results areconsistent with the data shown above and indicate that a preferentiallinkage of 2,6 sialic acid-galactose is involved in theanti-inflammatory activity of sialylated IgG.

Example 8 Removal of α2-6 but not 2,3 Sialic Acid Linkages Abrogates theImmunosuppressive Properties of IVIG

IVIG was treated with linkage specific sialidases (SAs), and thedigestion verified by lectin blotting (FIG. 3A). The top panel showspositive Sambucus nigra lectin (SNA) staining for α2-6 linkages in IVIG(left lane), and α2-3 SA tx IVIG (center lane), but not in α2-3,6 SA txIVIG (right lane). The middle panel is a dot blot for α2-3 sialic acidlinkages (MAL I), displaying positive staining for the fetuin positivecontrol only; 100 μg protein are loaded per dot. The bottom panel showscoomassie loading control. 10 μg/lane are shown in the blot and gel. Toexamine the effect of specific removal of sialic acid moieties, micewere given 1 g/kg of IVIG preparations prior to 200 μl of K/B×N sera. Asshown in FIG. 3B, footpad swelling was observed in mice administeredK/B×N sera (white circles) over the course of a week, as measured byclinical scoring. IVIG treated mice showed minimal swelling (blacktriangles), as did mice treated with α2-3 SA tx IVIG (white triangles),while mice receiving α2-3,6 SA tx IVIG (squares) were not protected fromfootpad swelling.

Example 9 Reduced Cytotoxicity does not Depend on the Nature of LinkageBetween Sialic Acid and Galactose

The inventors have previously demonstrated that sialylation of theN-linked glycan associated with the Fc domain of IgG resulted in reducedFcR binding, leading to a reduction in the A/I ratio (Kaneko, et al.,Science 313, 670 (2006)), a value derived from the affinity constantsfor an IgG Fc binding to individual activating (A) or inhibitory (I) IgGFc receptors. This ratio has been shown to be predictive of the in vivocytotoxicity for a specific IgG Fc (F. Nimmerjahn, J. V. Ravetch,Science 310, 1510 (2005)). Fc sialylation thus reduced the cytotoxicityof IgG antibodies in the induced thrombocytopenia model as well as in invitro models of ADCC (Kaneko, et al., Science 313, 670 (2006), Scallon,et al., Mol. Immunol 44, 1524 (2007)). The inventors, therefore, set outto determine if this reduction in FcR binding and cytotoxicity wasinfluenced by the sialic acid-galactose linkage. A monoclonalanti-platelet IgG2b antibody previously shown to lead to plateletconsumption was sialylated in vitro as described above and tested for invivo activity. Both terminal 2,3 and 2,6 in vitro sialylated IgG Fcreduced the cytotoxicity of this anti-platelet antibody, 6A6-IgG2b, inan in vivo model of thrombocytopenia (FIG. 4), consistent with previousstudies (Kaneko, et al., Science 313, 670 (2006), Scallon, et al., Mol.Immunol 44, 1524 (2007)). Thus, the effect of Fc sialylation on thecytotoxicity of an IgG antibody is not dependent on the specificity ofthe linkage to the penultimate galactose.

In contrast, the anti-inflammatory activity of the sialylated IgG Fcfragment (a property which the inventors have shown to be independent ofthe canonical IgG Fc receptors (F. Nimmerjahn, J. V. Ravetch, Science310, 1510 (2005); F. Nimmerjahn, J. V. Ravetch, J Exp Med 204, 11(2007)) displayed a clear preference for the 2,6 sialic acid-galactoselinkage, as seen in FIG. 3B.

These results further support the inventors' previous observations thatthe anti-inflammatory property of IVIG is mediated through a distinctpathway that does not involve binding to canonical FcγRs, which is insharp contrast to previously accepted models (Park-Min et al., Immunity26, 67 (2007); Siragam et al., Nat Med 12, 688 (2006)).

Example 10 In Vivo Anti-Inflammatory Activity of the 2,6 Sialylated IgGFc is Solely a Property of the IgG Fc Glycan

To fully demonstrate that the in vivo anti-inflammatory activity of the2,6 sialylated IgG Fc is solely a property of the IgG Fc glycan and notthe result of other components that might be found in the heterogeneous,IVIG Fc preparations, the anti-inflammatory activity of sialylated IVIGFc was recapitulated using a homogeneous, recombinant human IgG1 Fcsubstrate (rFc), derived from a cDNA (SEQ ID NO. 1) expressed in 293Tcells. The purified recombinant human IgG1 Fc fragment was glycanengineered in vitro, as described above, by β1,4 galactosylation,followed by 2,6 sialylation (FIG. 5A). The preparation was purified andcharacterized by lectin blotting and MALDI-TOF analysis (FIG. 5A) beforein vivo analysis. Glycosylation was confirmed by lectin blotting forterminal galactose with ECL (top panel), α2,6 sialic acid with SNA(middle panel), and coomassie loading controls are shown in the bottompanel.

Mice were administered IVIG, SNA+IVIG Fcs, or sialylated rFc (2,6ST rFc)1 hour prior to K/B×N sera, and footpad swelling was monitored over thenext several days. As seen in FIG. 5B, the 2,6 sialylated recombinanthuman IgG1 Fc fragment demonstrated comparable anti-inflammatoryactivity to that obtained with either IVIG-derived sialic-enriched Fcfragments (SNA+IVIG Fc) or in vitro 2,6 sialylated IVIG-derived Fcfragments (2,6ST IVIG Fc). Mean and standard deviation of clinicalscores of 4-5 mice per group are plotted; *denotes p<0.05 as determinedby Kruskal-Wallis Anova followed by Dunn's post hoc.

Each of these preparations was active at 30 mg/kg, as compared to the1,000-2,000 mg/kg required for native IVIG (Table 1).

TABLE 1 Different dosages of Fc fragment containing preparations resultin the same extent of inflammation suppression in arthritis model. IVIGprep IVIG IVIG Fc SNA⁺ IVIG SNA⁺ IVIG Fc 2,3ST IVIG Fc 2,6ST IVIG Fc2,6ST rFc Dose 1 g/kg 0.33 g/kg 0.1 g/kg 0.033 g/kg 0.033 g/kg 0.033g/kg 0.033 g/kg Amount/ 20 mg 6.66 mg 2 mg 0.66 mg 0.66 mg 0.66 mg 0.66mg mouse injection

Example 11 In Vivo Activity of IgG Subclasses Dependant on FcγRSpecificity

To address the role of individual FcγRs to the in vivo activities ofspecific IgG subclasses a series of antibodies were constructed for twodefined epitopes, in which the V_(H) regions of the cloned hybridomarecognizing either the melanosome gp75 antigen (TA99 family) oranti-platelet integrin antigen (6A6 family) were grafted onto theC57BL/6-derived G1, 2a, 2b or 3 constant regions and co-expressed withthe appropriate light chains in 293 T cells (Nimmerjahn et al., Immunity23, 41-51 (2005); Vijayasaradhi et al., J. Exp Med 171, 1375-80 (1990);and Clynes et al., Proc Natl Acad Sci USA 95, 652-6 (1998)). Theserecombinant antibodies were purified and tested for binding affinity totheir cognate antigen (Table 2) and to soluble, recombinantly expressedFcγR I, II, III or IV by surface plasmon resonance or to transfectedcells expressing a heterologous Fc receptor. Switching IgG constantregions did not affect the binding affinity of the resultant antibodiesto their respective antigens (Table 2).

TABLE 2 Affinities of TA99-antibody switch variants for gp75 KA(1/M)KD(M) TA99-IgG1  2.7 × 10⁹* 3.8 × 10⁻¹⁰ TA99-IgG2a 1.6 × 10⁹ 6.1 × 10⁻¹⁰TA99-IgG2b 1.8 × 10⁹ 5.7 × 10⁻¹⁰ TA99-IgG3 1.5 × 10⁹ 6.6 × 10⁻¹⁰*Antibody affinities were determined by surface plasmon resonance (SPR)analysis (Nimmerjahn et al., (2005)). A soluble version of theextracellular domain of gp75 was injected at a flow rate of 30 μl/minover the immobilized antibody variants and association and dissociationconstants were calculated. Each data point represents the mean of fiveexperiments performed in duplicates at different concentrations with aSE below 5%. Id.

In contrast, specific differences in binding affinity of each subclassto specific FcγRs were observed, as shown in Table 3. For example, IgG1bound with 10-fold higher affinity to the inhibitory receptor FcγRIIBthan to its activation counterpart, FcγRIII, while IgG2a and 2bdisplayed the reverse pattern, binding with 10-fold higher affinity forthe activation receptor FcγRIV than to the inhibitory receptor FcγRIIB.IgG3 did not bind to any of the known FcγRs. The ratio of activation toinhibitory binding (A/I), as shown in Table 2, thus can differ by asmuch as 2 orders of magnitude between IgG subclasses and FcRs.

TABLE 3 Affinities of Fcγ-receptors for antibody isotypes sFcγRIIBsFcγRIII sFcγRIV sFcγRI A/I IgG1(+fucose) 3.33 × 10⁶ 0.31 × 10⁶ −/− −/− 0.1 IgG1(−fucose) 1.32 × 10⁶ 0.51 × 10⁶ −/− −/−  0.4 IgG1(−SA) 4.00 ×10⁶ 0.50 × 10⁶ −/− −/−  0.1 IgG1(+SA) 0.39 × 10⁶ 0.07 × 10⁶ −/− −/−  0.2IgG2a(+fucose) 0.42 × 10⁶ 0.68 × 10⁶ 2.9 × 10⁷ 1.6 × 10⁸ 69**IgG2a(−fucose) 3.34 × 10⁶ 1.54 × 10⁶ 3.06 × 10⁸  1.8 × 10⁸ 92**IgG2b(+fucose) 2.23 × 10⁶ 0.64 × 10⁶ 1.7 × 10⁷ −/−  7** IgG2b(−fucose)1.0 × 10⁷ 1.06 × 10⁶ 2.03 × 10⁸  −/− 20** IgG3 −/− −/− −/− −/− −/− *Numbers represent the affinity (K_(A)) of the indicated antibodyisotypes to the indicated soluble Fcγ-receptors (sFcγR) as measured bysurface plasmon resonance analysis (Nimmerjahn et al., (2005)). A/I isthe ratio of the affinity of the activating (sFcγRIII or sFcγRIV,respectively) to the inhibitory receptor FcγRIIB. A double asteriskindicates the ratio of sFcγRIV to sFcγRIIB; −/− indicates no detectablebinding. Each data point represents the mean of five experimentsperformed in duplicates at different concentrations with a SE below 5%.+/− SA indicates antibodies enriched or depleted for sialic acid sugarresidues. Id.

Materials and Methods:

Mice: C57BL/6 and C57BL/6-129SF2/J mice were obtained from the JacksonLaboratory (Bar Harbor, Me.). γ^(−/−), FcγRIIB^(−/−) and FcγRIII^(−/−)mice were generated in our laboratory and backcrossed for 12 generationsto the C57BL/6 background. FcγRI^(−/−)129/B6 mice were generouslyprovided by Dr. Hogarth (The Austin Research Institute, Victoria,Australia). FcγRI/III^(−/−) mice were generated in our laboratory bycrossing FcγRI−/− with FcγRIII−/− mice and subsequent selection fordouble knockout animals. CR2−/−, C3−/− and C4−/− knockout mice wereprovided by Michael Carroll (CBR Institute for Biomedical Research,Harvard Medical School). Female mice at 2 to 4 months of age were usedfor all experiments and maintained at the Rockefeller University animalfacility. All experiments were done in compliance with federal laws andinstitutional guidelines and have been approved by the RockefellerUniversity (New York, N.Y.).

Cell culture: 293T, CHO-K1, B16-F10 and YB2/0 cells were culturedaccording to ATCC guidelines.

Antibodies and recombinant proteins: The 6A6 and TA99 antibody isotypeswitch variants and soluble Fcγ-receptors and gp75 were produced bytransient transfection of 293T cells and subsequent purification fromculture supernatants as described (Nimmerjahn, F., et al., (2005)). Forgeneration of TA99 antibody variants lacking fucose YB2/0 cells werestably transfected with the respective TA99 heavy and light chains.Fucose content of antibodies was verified by immunoblotting withbiotinylated aleuria aurantia lectin (Vector laboratories) followed bydetection with streptavidin-AP (Roche). Antibodies enriched or depletedfor sialic acid residues were generated by affinity chromatography withsambucus nigra lectin (Vector laboratories). Sialic acid content wasverified by immunoblotting with biotinylated sambucus nigra lectin(Vector laboratories). Purified C1q was from Calbiochem. TheFcγRIV-blocking antibody 9E9 has been described before Id. A hamsterIgG1 anti-TNP antibody was used as an isotype control antibody(Pharmingen).

Surface plasmon resonance (SPR) analysis: A Biacore 3000 biosensorsystem was used to assay the interaction of soluble mouse Fcγ-receptorsI, II, III and IV and soluble gp75 with the indicated antibody isotypes.Additionally, soluble human Fcγ-receptors IIA (131H-allele), IIB andIIIA (158F-allele) were used to measure the affinity to human IgGantibody isotypes. Antibodies or BSA as a control protein wereimmobilized at high and low densities to flow cells of CM5 sensor chips(Biacore) by standard amine coupling as suggested by the manufacturer.Soluble Fcγ-receptors were injected at 5 different concentrationsthrough flow cells at room temperature in HBS-EP running buffer (10 mMHepes, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20) at aflow rate of 30 μl/min. Soluble Fc-receptors were injected for 3 minutesand dissociation of bound molecules was observed for 10 minutes.Background binding to control flow cells was subtracted automatically.Control experiments were performed to exclude mass transportlimitations. Affinity constants were derived from sensorgram data usingsimultaneous fitting to the association and dissociation phases andglobal fitting to all curves in the set. As described for soluble humanFc-receptors a 1:1 Langmuir binding model closely fitted the observedsensorgram data and was used in all experiments Id. Alternatively,soluble Fc-receptors were immobilized to sensor chips with the sameresult described above.

In vivo model systems: The platelet depletion model: Experiments wereperformed essentially as described before. Id. Briefly, mice wereinjected intravenously with 4 μg of the recombinant 6A6 antibody isotypeswitch-variants diluted in 200 μl of PBS. Alternatively, mice wereinjected with 2 μg of the 6A6 antibody variants to studyFcγRIIB-mediated negative regulation of antibody functions in vivo.Platelet counts before injection and at indicated time points afterinjection were determined by blood collection (40 μl) from theretro-orbital plexus and measuring platelet counts of a 1:10 dilution inPBS/5% BSA in an Advia 120 haematology system (Bayer). To block FcγRIVin vivo mice were injected 30 minutes before administration of the 6A6antibody variants with 200 μg of the blocking FcγRIV antibody 9E9 orwith 200 μg of a hamster isotype control antibody (Pharmingen).

The B16-F10 lung metastasis model: Experiments were performed asdescribed (Vijayasaradhi et al., J Exp Med 171, 1375-80. (1990); andClynes et al., Proc Natl Acad Sci USA 95, 652-6. (1998)) with minormodifications. Mice were injected with 5×10⁵ B16-F10 tumor cellsintravenously and either left untreated or were injected with theindicated amounts of isotype control (Sigma) or TA99-isotype switchvariants on days 0, 2, 4, 7, 9, 11 intraperitoneally. To block FcγRIV invivo mice were injected with 200 μg of the 9E9 or the respective hamsterisotype control antibody on days 0, 2, and 4 intravenously. On day 15after tumour cell injection mice were sacrificed and lungs were analyzedfor the presence of surface metastasis by an investigator blinded forthe experimental setup.

Statistical analysis: The paired Student's t-test was used fordetermining significance of the results.

Example 12 A/I Ratios of Antibody Variants Predictive of In VivoBiological Activity

To determine how these differences in binding affinities relate to invivo biological activity, the ability of these antibodies to mediatetumor clearance or platelet depletion was investigated. As seen in FIG.6, both TA99 (FIGS. 6A and B) and 6A6 (FIGS. 6C and D) with IgG2aconstant regions display enhanced tumor or platelet clearance,respectively, as compared to these antibodies with IgG1 constantregions. IgG2a and 2b are equivalent in their ability to mediateplatelet clearance, while IgG2a results in enhanced tumor ADCC in themetastatic melanoma model as compared to IgG2b. The hierarchy ofactivity for the IgG subclasses is thus IgG2a≧IgG2b>IgG1>>IgG3. Themechanism of this differential activity was determined by repeatingthese experiments in specific activating FcγR or complement deficientstrains. No differences in in vivo activity were observed for IgG1, 2aor 2b in complement deficient strains (C4, C3 or CR1/2) (FIG. 7). Incontrast, IgG1, 2a and 2b were all dependent on activating FcγRexpression, since activity was abrogated in the common γ chain deficientbackground (FIGS. 8A, B and E). While IgG2a activity could result fromits ability to bind with high affinity to FcγRI, intermediate affinityto FcγRIV or low affinity to FcγR III, only FcγRIV binding was relevantto its in vivo activity (FIGS. 8C, D and E). Similarly, IgG2b activitywas FcγRIV dependent and FcγRIII independent (FIG. 8E). In contrast,IgG1 mediated effector activity was exclusively FcγRIII dependent (FIG.8E).

The balance of activation to inhibitory receptor expression has beenshown to determine the threshold for IgG mediated effector celltriggering (Ravetch and Lanier, Science 290, 84-9 (2000)). The bindingaffinities of IgG subclasses to the inhibitory receptor vary by a factorof 10, suggesting that a differential dependence of the subclasses onthe inhibitory effect of FcγRIIB might be observed. As seen in FIG. 9,IgG1 displays the greatest enhancement in activity in mice lacking theinhibitory receptor in both the tumor clearance and platelet depletionmodels (FIG. 9A-C), while IgG2a shows the smallest enhancement in bothmodels. The magnitude of IgG2b enhancement in FcγRIIB deficient strainsdiffers in the two models, showing significant enhancement in the tumorclearance model and minimal enhancement in the platelet depletion model.This difference is likely due to the intermediate A/I ratio of thisreceptor rendering it more sensitive to the levels of surface expressionof FcγRIIB on the specific effector cells mediating the in vivoresponses. Since different populations of effector cells are responsiblefor the biological responses in the two models, the IgG2b data supportour previous observations that RIIB levels are minimal on splenicmacrophages, the cell type responsible for platelet clearance(Nimmerjahn et al., Immunity 23, 41-51. (2005); and Samuelsson et al.,Science 291, 484-6. (2001)), and higher on alveolar macrophages, therelevant effector cells in the metastatic melanoma model (Shushakova etal., J Clin Invest 110, 1823-30 (2002)). These results demonstrate thepredictive value of the A/I ratio in determining the contribution ofinhibitory signaling to in vivo activities. A high A/I ratio, as foundfor IgG2a, renders the antibody essentially insensitive to differencesin FcγRIIB expression on different effector cell populations, while alow A/I ratio, as found for IgG1, maximizes the role of FcγRIIB. Forantibodies with intermediate A/I ratios, like IgG2b, the in vivoactivity will be determined by the specific effector cell involved inthe response, reflecting the differences in FcγRIIB levels and itsregulation by the cytokine milieu. This difference in FcγRIIB dependencefor IgG1 and IgG2 may reflect the biological roles of these subclassesin vivo, insuring that the most abundant subclass, IgG1, is under tightregulation by an inhibitory receptor, thus preventing effector cellactivation in the absence of a second signal that down regulates FcγRIIBand lowers the threshold for activation. Such second signals areprovided by pro- and anti-inflammatory cytokines and chemokines whichhave been demonstrated to alter the levels of surface expression ofactivation or inhibitory receptors (Shushakova (2002)). In contrast, therole of inhibitory receptor expression on IgG2a potency, and to a lesserextent, IgG2b, is less significant, reflecting the effector bias ofT_(H1) cytokines which induce both IgG2a switching and FcγRIVexpression.

Example 13 Modified Antibody with a Lower Amount of Fucose and a GreaterA/I Ratio Compared to Unmodified Antibody

The relationship between the A/I ratio of IgG subclasses and in vivoactivity was further tested using modified IgG constant regions. FcRbinding to IgG is dependent on the presence of N-linked glycosylation atposition 297; deglycosylation abrogates all FcR binding (Krapp, J MolBiol 325, 979-89 (2003)). However, selective removal of specificcarbohydrates, such as fucose, has been suggested to modify human IgG1binding to human FcγRIII and thus to NK cell mediated ADCC in vitro(Shields, R. L., et al., J Biol Chem 277, 26733-40. (2002); T. Shinkawaet al., J Biol Chem 278, 3466-73 (2003); and Niwa et al., Cancer Res 64,2127-33 (2004)). Fucose-deficient TA99-IgG1, 2a and 2b were prepared andtheir binding to FcγRI, II, III and IV was compared. C1q or antigenbinding was not affected by the lack of fucose as described before(Shields (2002)). However, as shown in FIG. 10 and Table 3, fucosedeficient antibodies differed in their binding affinities to theircognate FcγRs, with TA99-IgG1 with or without fucose displaying minimaldifferences in binding to FcRIIB and III, while IgG2a and 2b fucosedeficient antibodies bound with an order of magnitude higher affinity toFcRIIB and FcRIV as compared to fucose sufficient antibodies. Thesedifferences in binding affinities resulted in altered A/I ratios thatwere most pronounced for IgG2b (FIG. 10 and Table 3) and translated intosignificantly enhanced in vivo activity for IgG2b. This selective effectof de-fucosylation on FcR binding further illustrates the specificity ofIgG subclasses in their interactions with individual FcRs and thepredictive value of the A/I ratio in determining in vivo activity.

Example 14 Modified Antibody with a Lower Amount of Sialic Acid

The role of sialic acid residues in antibody sugar side chains wasinvestigated by injecting mice with 6A6-IgG1 antibody variants enrichedor depleted for sialic acid residues and measuring antibody mediatedplatelet depletion. Antibodies enriched for sialic acid in their sugarside chains displayed strongly reduced affinity for both the activatingFcRIII and inhibitory FcRIIB (Table 3). Consistent with this loss inoverall affinity for Fc-receptors and the low A/I ratio of 0.18, thisantibody had a severely impaired in vivo activity and mediated onlyminimal platelet depletion (FIG. 11).

Example 15 Human Antibodies Display Differential A/I Ratios

To investigate if human antibody isotypes also display differential A/Iratios soluble versions of human Fcγ-receptors were prepared and theiraffinity for human IgG antibody isotypes was measured by surface plasmonresonance analysis. As shown in Table 4 human FcRs also havedifferential A/I ratios for individual human IgG antibody isotypes.

TABLE 4 Affinities of Human FcRs to Human IgG Isotypes A/I sFcRIIBsFcRIIA sFcRIIIA IIA/IIB IIIA/IIB IgG1 7.8 × 10⁴ 2.5 × 10⁵ 4.3 × 10⁵ 3.15.5 IgG1-fucose 7.8 × 10⁴ 2.4 × 10⁵ 6.0 × 10⁶ 3.1 76 IgG2 3.0 × 10⁴ 1.4× 10⁵ 1.4 × 10⁴ 4.5 0.4 IgG2-fucose 2.6 × 10⁴ 1.3 × 10⁵ 1.4 × 10⁵ 5.35.7 IgG4 4.8 × 10⁴ 3.5 × 10⁴ −/− 0.7 n.a. * Numbers represent theaffinity (K_(A)) of the indicated antibody isotypes to the indicatedsoluble Fcγ-receptors (sFcγR) as measured by surface plasmon resonanceanalysis (Nimmerjahn et al., (2005)). A/I is the ratio of the affinityof the indicated activating to the inhibitory receptor FcγRIIB. −/−indicates no detectable binding; n.a. indicated not applicable. Eachdata point represents the mean of five experiments performed induplicates at different concentrations with a SE below 5%.

In contrast to the single chain inhibitory Fc-receptor, activating FcγRs(with the exception of human FcγRIIA) cannot transmit activating signalsin the absence of an accessory chain, the common gamma chain (γ-chain),that carries an ITAM motif required for triggering cell activation.FcγRI, FcγRIII and FcγRIV are dependent on γ-chain expression; thusdeletion of this receptor subunit leads to the functional loss of allactivating Fc-receptors and several other non-FcR-related proteins suchas PIR-A and NK cell cytotoxicity receptors (Moretta et al., Annu RevImmunol 19, 197-223 (2001); Ravetch, (2003)).

The only IgG isotype that could consistently be assigned to anindividual activating Fc-receptor in vivo was IgG1. The deletion of thelow affinity receptor FcγRIII abrogates IgG1 mediated effector functionsin various models like arthritis, glomerulonephritis, IgG-dependentanaphylaxis, IgG mediated hemolytic anemia and immunothrombocytopenia(Hazenbos et al., Immunity 5, 181-188 (1996); Meyer et al., Blood 92,3997-4002 (1998); Fossati-Jimack et al., J Exp Med 191, 1293-1302(2000); Ji et al., Immunity 16, 157-168 (2002); Bruhns et al., Immunity18, 573-581 (2003); Fuji et at., Kidney Int 64, 1406-1416 (2003);Nimmerjahn et al., (2005)). Under many circumstances, such as hostresponse to viral or bacterial infections (Coutelier et al., J Exp Med165, 64-69 (1987); Schlageter and Kozel, Infect Immun 58, 1914-1918(1990); Markine-Gorianyoff and Coutelier, J Virol 76, 432-435 (2002);Taborda et al, J Immunol 170, 3621-3630 (2003)), and antibody-mediatedcytotoxicity or antibody-based therapy (Kipps et al., J Exp Med 161,1-17 (1985); Fossati-Jimack et al., J Exp Med 191, 1293-1302 (2000);Uchida et al., J Exp Med 199, 1659-1669 (2004); Nimmerjahn et al.,(2005)) the most potent antibody isotypes are of the IgG2a and IgG2bisotype. Therefore, a thorough understanding of how these isotypes exerttheir function is essential.

Considering the isotype specificities of the high affinity FcγRI(binding exclusively IgG2a) and the low affinity FcγRIII (binding IgG1,IgG2a, and IgG2b) (reviewed in Ravetch and Kinet, Annu Rev Immunol 9,457-492 (1991); Hulett and Hogarth, Adv Immunol 57, 1-127 (1994)), thesetwo receptors are likely candidates responsible for IgG2a and IgG2beffector functions. Although there is some suggestion that FcγRI and IIImight participate in a limited fashion in IgG2a-mediated effectorresponses (Ioan-Facsinay et al., Immunity 16, 391-402 (2002); Barnes etal., Immunity 16, 379-389, (2002)), the majority of studies concludedthat IgG2a and IgG2b triggered effects occur independently of these tworeceptors, but in a gamma chain dependent manner (Hazenbos et al.,Immunity 5, 181-188 (1996); Meyer et al., (1998); Fossati-Jimack et al.,(2000); Uchida et al., (2004); Nimmerjahn et al., (2005)). Especially inthe case of IgG2a these results seem to be surprising as FcγRI shows ahigh affinity for this isotype (KA: 10⁸-10⁹ M⁻¹). However, the increasedaffinity allowed this receptor to bind monomeric IgG2a as efficiently asimmune complexes (ICs), indicating that newly generated ICs would beexpected to have only limited access to FcγRI (FIG. 12A).

FcγRIV requires γ chain for its surface expression (Nimmerjahn, (2005))and, as has been described for other γ-chain dependent Fc-receptors,cross-linking of FcγRIV by immune complexes induces activating signalingpathways leading to sustained calcium flux (reviewed in Ravetch andBolland, (2001); Nimmerjahn et al., (2005)).

Even if several activating Fc-receptors with the same isotypespecificity are present on the same cell, only those Fc-receptors willbe engaged that show the optimal affinity for the respective isotype(FIG. 12A). Therefore, IgG1 immune complexes will only trigger FcγRIIIas it is the only activating Fc-receptor that can bind IgG1 (Takai,(1994); Hazenbos et al., (1996); Meyer et al., (1998); Nimmerjahn etal., (2005)). IgG2a and IgG2b, despite their ability to bind FcγRI (inthe case of IgG2a) or FcγRIII (in the case of IgG2a and 2b) willfunctionally be dependent on FcγRIV, as FcγRI will be occupied bymonomeric IgG2a and the low affinity of RIII will not result inproductive engagement at normal serum concentration of these isotypes.These same principles also apply for the human system, where it has beenshown that human FcγRIIIA has a higher affinity for IgG1 as compared tohuman FcγRIIA. In addition, the presence of allelic variants which showdifferential affinities for the specific antibody isotypes furthersupports this concept (Dijstelbloem et al., Trends Immunol, 22, 510-516(2001)).

The present invention provides a mechanistic basis for the observedvariation in IgG subclass activity in both active and passivevaccination and in the variable pathogenicity of the IgG subclasses inautoimmune conditions. The selective FcγR binding affinities of the IgGsubclasses, and not their ability to fix complement, is predictive ofthe in vivo activity for cytotoxic antibodies in models of tumorclearance, platelet and B cell depletion (Uchida et al., J Exp Med 199,1659-69. (2004); Clynes and Ravetch (1995); Clynes (1998); Samuelsson(2001)). Similarly, the biological consequences of modifications to IgGantibodies are, in turn, dependent on their effects on specific FcRbinding affinities that result in changes to the ratio of activation toinhibitory receptor affinities. These considerations will be significantfactors in the design of both antibody-based immunotherapeutics andactive vaccination protocols to insure either the selective engineeringof IgG Fc domains or induction of IgG subclasses with optimal FcγRactivation to inhibitory ratios.

All patent and non-patent publications cited in this disclosure areincorporated herein in to the extent as if each of those patent andnon-patent publications was incorporated herein by reference in itsentirety. Further, even though the invention herein has been describedwith reference to particular examples and embodiments, it is to beunderstood that these examples and embodiments are merely illustrativeof the principles and applications of the present invention. It istherefore to be understood that numerous modifications may be made tothe illustrative embodiments and that other arrangements may be devisedwithout departing from the spirit and scope of the present invention asdefined by the following claims.

What is claimed is:
 1. An isolated polypeptide containing at least oneIgG Fc region, having altered properties compared to an unpurifiedantibody preparation, wherein sialylation of the isolated polypeptide ishigher than the sialylation of the unpurified antibody preparation. 2.The isolated polypeptide of claim 1, wherein said at least one IgG Fcregion is glycosylated with at least one galactose moiety connected to arespective terminal sialic acid moiety by a α2,6 linkage, and whereinsaid polypeptide having a higher anti-inflammatory activity as comparedto an unpurified antibody preparation.
 3. The isolated polypeptide ofclaim 1, wherein said at least one IgG Fc region is glycosylated with atleast one galactose moiety connected to a respective terminal sialicacid moiety by a α2,6 linkage, and wherein said polypeptide having areduced binding to an Fc activating receptor selected from the groupconsisting of Fc?RIIA, Fc?RIIC and Fc?RIIIA, as compared to anunpurified antibody preparation.
 4. The isolated polypeptide of claim 1comprising a human IgG1, IgG2, IgG3 or IgG4 Fc region, said polypeptidehaving a higher content of the at least one galactose moiety connectedto the respective terminal sialic acid moiety by a α2,6 linkage ascompared to an unpurified antibody.
 5. The isolated polypeptide of claim1, derived either from a naturally occurring antibody source or arecombinant antibody source.
 6. The isolated polypeptide of claim 1,wherein said unmodified antibody comprises IVIG.
 7. The isolatedpolypeptide of claim 1 produced from a recombinant source and lackingFab region, wherein said at least one IgG Fc region is glycosylated withtwo galactose moieties.
 8. The isolated polypeptide of claim 1 encodedby a nucleic acid sequence comprising SEQ ID NO:
 1. 9. The isolatedpolypeptide of claim 1, derived from a cell line having an enhancedactivity of creating α2,6 linkages between at least one galactose moietyand a respective terminal sialic acid in a protein's polysaccharidechain.
 10. The isolated polypeptide of claim 1, modified by treatmentwith α2-6 sialyltransferase.
 11. A method of modulating properties of apolypeptide comprising an Fc region comprising altering the sialylationof the polysaccharide chain of the Fc region.
 12. A method of claim 11,wherein said properties comprise a higher anti-inflammatory activitythan an unpurified antibody.
 13. The method of claim 11, wherein thestep of altering sialylation comprises: providing an unpurified sourceof the polypeptide containing at least one Fc region, said unpurifiedsource of the polypeptide containing at least one Fc region comprising aplurality of the polypeptides containing at least one Fc region having apolysaccharide chain comprising a terminal sialic acid connected to agalactose moiety through a α2,6 linkage, and a plurality of thepolypeptides containing at least one Fc region lacking a polysaccharidechain comprising a terminal sialic acid connected to a galactose moietythrough the α2,6 linkage; and increasing the ratio of the plurality ofthe polypeptides containing at least one Fc region having thepolysaccharide chain comprising the terminal sialic acid connected tothe galactose moiety through the α2,6 linkage to the plurality of thepolypeptide containing at least one Fc region lacking the polysaccharidechain comprising the terminal sialic acid connected to the galactosemoiety through the α2,6 linkage.
 14. The method of claim 11, wherein theunpurified source of the polypeptide containing at least one Fc regionis provided from expressing a vector comprising a nucleic acid sequencein an expression system, wherein said nucleic acid sequence istranslated into an IgG antibody.
 15. The method of claim 11, wherein thestep of increasing the ratio of the plurality of the polypeptidescontaining at least one Fc region having the polysaccharide chaincomprising the terminal sialic acid connected to the galactose moietythrough the α2,6 linkage to the plurality of the polypeptide containingat least one Fc region lacking the polysaccharide chain comprising theterminal sialic acid connected to the galactose moiety through the α2,6linkage is achieved through a removal of the polypeptides containing atleast one Fc region lacking the polysaccharide chain comprising theterminal sialic acid connected to the galactose moiety through the α2,6linkage.
 16. The method of claim 15 wherein said removal is achieved bya method selected from the group consisting of HPLC, lectin affinitychromatography, high pH anion exchange chromatography, and anycombination thereof.
 17. The method of claim 16, wherein the lectinaffinity chromatography is performed using a lectin having a loweraffinity to α2,6 linkages than to α2,3 linkages between the galactosemoiety and the terminal sialic acid.
 18. The method of claim 15, whereinthe step of increasing the ratio of the plurality of the polypeptidescontaining at least one Fc region having the polysaccharide chaincomprising the terminal sialic acid connected to the galactose moietythrough the α2,6 linkage to the plurality of the polypeptide containingat least one Fc region lacking the polysaccharide chain comprising theterminal sialic acid connected to the galactose moiety through the α2,6linkage is achieved through an enrichment of said unpurified source ofthe polypeptide containing at least one Fc region having thepolysaccharide chain comprising the terminal sialic acid connected tothe galactose moiety through the α2,6 linkage.
 19. The method of claim18 wherein said enrichment is achieved by a method selected from thegroup consisting of HPLC, lectin affinity chromatography, high pH anionexchange chromatography, and any combination thereof.
 20. The method ofclaim 19, wherein the lectin affinity chromatography is performed usinga lectin having a higher affinity to α2,6 linkages than to α2,3 linkagesbetween the galactose moiety and the terminal sialic acid.
 21. Themethod of claim 18, wherein said enrichment is achieved by a chemicalreaction with an enzyme creating α2,6 linkages between the carbohydrateattached to the polypeptide containing least one Fc region and aterminal sialic acid.
 22. A method of treating an inflammatory diseaseselected from the group consisting of arthritis, thrombocytopenia, andnephritis comprising administering to a patient a therapeuticallyeffective dose of the polypeptide of claim
 1. 23. A method of treatingan inflammatory disease comprising administering to a subject in needthereof a therapeutic composition comprising a plurality of isolatedpolypeptides, each containing at least one IgG Fc region, wherein afirst portion of the respective Fc regions comprises respectivecarbohydrate chains having galactose moieties connected to respectiveterminal sialic acid moieties by 2,6 linkage; a dose of the therapeuticcomposition is smaller than a dose of a second composition whichcomprises a plurality of isolated polypeptides, each containing at leastone IgG Fc region, having a second portion of the respective Fc regionscomprising respective carbohydrate chains having galactose moietiesconnected to respective terminal sialic acid moieties by 2,6 linkage;and either the first portion is greater than the second portion, wherebythe dose of the therapeutic composition and the dose of the secondcomposition suppress inflammation to substantially the same extent, orthe first portion is greater than the second portion, whereby thetherapeutic composition suppresses inflammation to substantially agreater extent than an equal dose of the second composition.
 24. Acomposition comprising glycoproteins containing an Fc region wherein thecomposition has been formulated to contain sialylated glycoproteins inan amount sufficient to achieve an immunosuppressive activity in amammal.
 25. The composition of claim 24, wherein the compositioncomprises sialylated glycoproteins in an amount of about 5% or more. 26.The composition of claim 24, wherein the composition comprisessialylated glycoproteins in an amount of about 10% or more.
 27. Thecomposition of claim 24, wherein the composition comprises sialylatedglycoproteins in an amount of about 30% or more.
 28. The composition ofclaim 24, wherein the composition comprises sialylated glycoproteins inan amount of about 5% to about 30%.
 29. The composition of claim 24,wherein the sialylated glycoproteins comprise one or more terminalsialic acid residues or analogues thereof.
 30. The composition of claim29, wherein the terminal sialic acid residue(s) is linked to theglycoprotein by an alpha 2,6 linkage.
 31. An IVIG derived compositionformulated to contain sialylated Fc containing glycoproteins in anamount of about 5% to about 30% and wherein the sialylated glycoproteinscomprise one or more terminal sialic acid residues linked to theglycoprotein by an alpha 2,6 linkage.
 32. A recombinant Fc glycoprotein,or fragment thereof, comprising at least one terminal sialic acidresidue(s), or analogue(s) thereof, linked to the glycoprotein by analpha 2,6 linkage.
 33. A recombinant Fc glycoprotein comprising anN-linked carbohydrate at Asn 297 wherein in the carbohydrate has abiantennary GlnNac2, Man3, GlcNAc2, Gal2 structure having one or moreterminal sialic acid residue(s) linked by an alpha 2,6 linkage.
 34. AnFc containing glycoprotein of any of the above claims wherein the Fcregion is IgG or a subclass thereof.
 35. A pharmaceutical preparationcomprising the glycoproteins of claim
 24. 36. A method of treating aninflammatory disorder in a subject using the pharmaceutical preparationof claim 35.